Survey and Molecular Characterization of Trichomonads in Pigs in Anhui Province, East China, 2014.

Background: In pigs, several different trichomonad species such as Tritrichomonas foetus, Tetratrichomonas buttreyi, and Pentatrichomonas hominis have been described as inhabiting the digestive tract. However, little information is available on the epidemiology of these neglected parasites in the Chinese pig population. Methods: The prevalence of T. suis, T. buttreyi and P. hominis among 500 fecal specimens from pigs at seven pigs farms in Anhui Province in China between Oct and Dec 2014, was determined by PCR and DNA sequence analysis of the small subunit ribosomal RNA (SSU rRNA) genes. Results: The prevalence rates for T. suis, T. buttreyi, and P. hominis were 2.8% (14/500), 42.0% (210/500) and 7.8% (39/500), respectively. Mixed infections of two or three trichomonads were detected in 24 samples. The prevalence of the three trichomonads differed significantly between some age groups, with higher infection rates of T. suis and T. buttreyi in nursery pigs and P. hominis in preweaned pigs. The SSU rRNA sequences from T. suis and P. hominis showed 100% homology with their respective homologous database sequences. However, we observed minor allelic variations in the SSU rRNA sequences from T. buttreyi, and the five representative sequences identified were named firstly as types 1, 2, 3, 4 and 5. Moreover, type 1 was found to be dominant in the present study. Conclusion: These findings highlight the potential risk posed by pigs in the transmission of trichomonad infections to humans and other animals.


Introduction
arasites of the Tritrichomonas genus, which consists of commensal and pathogenic species from a broad host range of vertebrates and invertebrates, are frequently encountered in veterinary medicine (1)(2)(3)(4). They are characterized by the presence of three-to-five anterior flagella and a single recurrent flagellum that functions as an undulating membrane (5). The following three different trichomonad species were originally reported as pathogenic protists in pigs: Tritrichomonas suis, T. rotunda, and Tetratrichomonas buttreyi (6). Hypotrichomonas acosta and Trichomitus batrachorum, previously isolated from squamate reptiles and a frog, respectively, have also been described in pigs (7). Recently, our laboratory reported the occurrence of Pentatrichomonas hominis in pigs and 24.1% (38/158) of the pigs in Changchun, China tested positive for this species (4,8).
Among the above mentioned porcine trichomonad species, T. suis is the well-studied and has been reclassified as a synonym of the parasitic trichomonad responsible for bovine and feline trichomonosis, T. foetus (9)(10)(11). T. suis was initially regarded commensalic and nonpathogenic in pigs (12). However, T. suis was a facultative pathogen of the pig large intestine, thus highlighting its medical importance (9)(10)13). T. buttreyi is found in pig and cattle fecal samples and is considered a commensal organism (10,14). Similarly, P. hominis is presumed to be a commensal organism that can overgrow opportunistically in hosts with diarrhea from other causes (2). However, P. hominis was described as a pathogen associated with gastrointestinal discomfort, respiratory tract infections and rheumatoid arthritis in humans (15)(16)(17). Additionally, P. hominis has the ability to propagate in the cecum of piglets and is most likely a pathogen in pigs with underlying comorbidities (4).
China is the world's largest producer and consumer of pork. Pig farming plays a particularly important role in animal husbandry in Anhui Province. The rapid growth in pig farming and the high human population density in this region may increase the spread of infectious micro-organisms carried in the feces of domestic animals (including trichomonads) to humans. Unfortunately, few published studies are available on the epidemiology of trichomonads in pigs in China. Recently, our laboratory reported the prevalence of trichomonads in pigs in Changchun city, Jilin Province, northeast China (8).
To date, no studies have been published for pigs in Anhui Province, China. In the present study, we conducted an extensive survey of pigs and accurately determined the prevalence of trichomonads using molecular methods. We also genetically characterized the trichomonads identified in this study and discussed the risk factors for trichomonad infections in pigs in Anhui Province, China.

Fecal specimen collections
Overall, 500 fresh fecal samples including those from preweaned pigs (under 30 d old), nursery pigs (30-60 d old), and growing pigs (over 60 d old) were collected from seven pig farms located in seven prefectures in Anhui Province between Oct and Dec 2014 (Fig. 1). All the fecal samples were stored at 4 °C prior to DNA extraction within three days.
The present experimental protocol was conducted in accordance with the strict guidelines and recommendations from the Guide for the Animal Care and Welfare Committee of Anhui Science and Technology University, China.

DNA extraction
Genomic DNA was extracted directly from a 200-mg sample of each fecal specimen using a Stool DNA Kit (Tiangen, Beijing, China), according to the manufacturer's instructions. The DNA was eluted in a 50 μl volume and stored at −20 °C until use.
The concentrations and reagents used for the secondary amplification were identical to those used in the primary PCR, except that 2 μl of the primary PCR product was substituted for genomic DNA. The secondary PCR was also amplified according to the conditions except that the denaturation duration (45 sec instead of 30 sec) and extension duration (45 sec instead of 30 sec) for T. suis and P. hominis were modified (4). All PCRs were performed in duplicate. Control samples without DNA (negative control) and with the three trichomonad species genomic DNA (positive control) were included in each PCR run.

DNA sequencing
Secondary PCR products were subjected to electrophoresis in 2% agarose gels and then visualized by ethidium bromide staining. PCR products were purified and the accuracy of the nucleotide sequences was confirmed by twodirectional sequencing. DNA sequences were aligned against reference sequences using Bi-oEdit v 7.1.3.0 software (Ibis Biosciences, Carlsbad, CA, USA). Neighbor-joining trees were constructed using MEGA 5 software (http://www.megasoftware.net/), and evolutionary distances were calculated using the Kimura 2-parameter model. The reliability of the cluster formation was evaluated by the bootstrap method with 1000 replicates. Representative SSU rRNA sequences from this study have been deposited in GenBank under accession number KX833160 (for T. suis), KX833155-KX833159 (for T. buttreyi), and KX833161 (for P. hominis).

Statistical analysis
The 2 test was used to compare the three trichomonad infection rates in the different groups of pigs, using SPSS for Windows (13.0 standard version; (Chicago, IL, USA). Differences were considered statistically significant at P<0.05.

Discussion
Traditionally, trichomonad infections are diagnosed through microscopic examination of fresh fecal samples or cultures of fecal samples. However, very often different trichomonad species that share similar morphological features cannot be differentiated by micro-scopic examination (18,19). Therefore, sensitive and specific molecular methods are now used widely to identify trichomonad species and strains (20)(21)(22). Although several porcine trichomonad species have been described, only a handful of epidemiological reports documenting naturally occurring infections of pigs with trichomonads around the world have been published, partly because of the relative lack of importance of these species in veterinary medicine (8,(23)(24)(25). In the present study, the prevalence of the three trichomonad species in pigs in Anhui, East China, was investigated by nested PCR targeting the SSU rRNA gene and genetic similarities among the three trichomonads in pigs and other hosts were also analyzed. We found that the overall infection rate for trichomonad species in the pigs, as determined by PCR, was 47.4% (237/500). This proportion is quite similar to the value (43.0%; 68/158) for northeast China (8), but lower than the value (52.1%; 100/192) from in situ hybridization and the value (77.4%; 24/31) from culture experiments (25). Besides, six trichomonad species have been identified in pigs, namely, T. suis, T. rotunda, and T. buttreyi, H. acosta, T. batrachorum and P. hominis (4,(6)(7)(8). Among these species, T. suis is the most common in various geographical locations of the world (25% in UK, 56.3% in Japan, 12.0% in China, 64.5% in Austria). In contrast, our study found a low prevalence (2.8%) of T. suis in pigs in East China. Indeed, we found that T. buttreyi (42.0%) was the most common species in the present study.
These results clearly differ from those reported for northeast China, where P. hominis (24.1 %) was predominant, followed by T. buttreyi (14.6%) and T. suis (12.0%) (8). The differing prevalences of the trichomonad species from the various studies may reflect possible variations in the samples and in the examination methods used, as well as age, sample sizes, seasonality, host health status, specimen collection timing, and geo-ecological conditions (26) .
In the present study, all the age groups were positive for three trichomonads, which is consistent with the results of a previous study in northeast China (8). Nevertheless, an agerelated difference was observed in the prevalence of the three trichomonads in this study. T. suis and T. buttreyi had significantly higher prevalence rates in nursery pigs than in preweaned and growing pigs (P<0.01), whereas preweaned pigs had a significantly higher infection rate for P. hominis than that of the nursery pigs (P<0.05). Contrastingly, no significant relationship was found for the presence of the three parasite species and the animal age in previous studies conducted in northeast China (8). The SSU rRNA sequences obtained from T. suis and P. hominis our study were 100% identical to the sequences obtained from other animal isolates, which is consistent with previous observations (8).
Interestingly, most T. suis isolates from pigs are the 'cattle genotype', while a few of them are the 'cat genotype', as determined mainly by the presence of a conserved single nucleotide polymorphism in the internal transcribed spacer-2 gene (24,25). In the present study, the genotypes of the T. suis isolates from pigs remain unresolved, and additional experimental studies are needed to obtain better genotyping.
Our study has also described minor allelic variation in T. buttreyi from East China, a result similar to that of a previous study in northeast China (8). The five representative sequences from the present study contained only three variable positions but allowed the identification of five sequence types (types 1 to 5). The minor genetic distinctions between the five types indicated that they were from the same T. buttreyi strain. Some sequences from different hosts currently available in GenBank displayed 100% identity to types 1 to 3 in this work, respectively, and the majority of them belonged to the type 2 classification according to our BLAST check and comparative analysis. Moreover, type 1 (51.0%) was found to be dominant in the present study, followed by type 2 (30.0%) and type 3 (11.0%), with a few pigs also infected with types 4 and 5.

Conclusion
Pigs can act as potential sources of trichomonad infections in humans and other animals. Because of the high frequency of human contact with pigs in China, further studies are needed to clarify the significance of pigs in the epidemiology of trichomonads in humans and other animals.